Transformation of Yeast

One of the most important techniques of modern molecular biology is the
ability to
introduce a specific gene into an organism and have that genetic
information expressed
by the organism. This process, called genetic transformation, played a
critical role in the
history of molecular biology and the discovery that DNA was the genetic
material. Four
scientists, Griffith in the 1920's and Avery, McCarty, and MacLeod in the
1940's,
showed that the apparent "transformation" of one bacterial type into
another required
DNA and no other biological molecule.

Today, transformation is not limited to bacteria, and in fact, is used
routinely by
molecular biologists to manipulate and study the behavior of genes.
"Knock-out" and transgenic mice are created through transformation of
embryonic stem cells. Transgenic plants expressing foreign genes have been
constructed through transformation procedures. The lab described below
demonstrates the properties of transformation using yeast and a simple
colony color gene. Yeast transformation involves several steps. After
completion of these steps, the yeast will express the new characteristics
or phenotype of
the added DNA.

This experiment uses a yeast strain which is defective in
an adenine synthesis gene, ADE1. The metabolic defect in the
mutants causes them to require
adenine for growth and to accumulate a red pigment which causes the
colonies to appear pink or red in color. When these ade1 mutant
yeast are transformed with the normal
ADE1 gene, they gain the ability to grow in the absence of adenine
and form normal cream colored colonies.
The DNA molecules used in this transformation system are plasmids, small,
circular
pieces of DNA. They behave in many ways like real chromosomes which carry
genes in
cells but plasmids are much more convenient. There are two types of
plasmids carrying
the yeast ADE1 gene available for use in this experiment. The
YEpADE1
plasmid (map)
exists in multiple copies in each transformed yeast cell; however, it is
unstable and can
be lost from transformed yeast at a relatively high frequency. The YCpADE1
plasmid (map)
exists in one copy in a haploid yeast cell, just like the normal
chromosomes, and since it
carries a yeast centromere, will be stably sorted into the daughter cells
each time the
yeast divides. Either plasmid can be used effectively to demonstrate the
phenomenon of
transformation.

Basic transformation protocol

Day One: Touch a sterile toothpick to the stock sample of yeast HA1
or HA1L and then make two or three streaks of the cells on a YED agar
plate. Incubate the
plate at room temperature or in a 30 C. incubator for 1-2
days. Alternatively, a liquid culture can be grown with shaking,
overnight.

Day Two:

1. Prepare yeast/LiAc/TE suspension.
Use the flat
end of a sterile toothpick to scrape about 2-3 centimeters of a yeast
streak from the
agar. Suspend the cells in 0.5 mL of LiAc/TE solution in a sterile
microcentrifuge tube.

2. Prepare DNA / carrier DNA tubes. To a new sterile microcentrifuge
tube,
add 21 microliters of
the plasmid DNA/carrier DNA mix.
You may wish to make some control tubes. Some examples of possible control
tubes are
1) carrier DNA only; 2) plasmid DNA only; 3) no DNA

3. Add yeast to DNA tubes. To the DNA tube(s) you
prepared add 0.2 mL per tube of the yeast/LiAc/TE suspension from step 1.
Discard the remainder of the yeast/LiAc/TE suspension.

5. Tightly close the tube and mix well by inverting the tube several
times.

6. Incubate this mixture at room temperature for approximately 20
minutes.

7. Heat shock the DNA/yeast/PLT suspension(s) for a minimum of 5
minutes (maximum
of 15 minutes) in a 42oC water bath.

8. Centrifuge the suspension to pellet the yeast cells. Discard the
supernatant (liquid solution) from the microfuge tube(s).

9. Resuspend the pelleted yeast cells by adding 1 mL of TE solution.
Tap gently, use a sterile toothpick or vortex to suspend the cells in the
TE solution.

10. Plate suspension on MV agar. From each yeast suspension tube use a
fresh sterile pipet to transfer 0.2 mL of yeast suspension to each of one
or two MV (selective medium) plates.
Use a fresh sterile spreader to spread the cells from each tube evenly
over the surface of the agar.

11. Incubate the plates for 3-5 days at room temperature or in a 30oC
incubator.

Day 5: Observe and record your results. For each
experimental condition count the number of colonies and record the yeast
colony colors.

Plasmid Loss: An Extension of the Transformation Exercise

Transformed cells containing the unstable YEpADE1 plasmid will maintain
the white
phenotype only under selective pressure (the absence of adenine in their
growth medium).
In these conditions, cells which do not contain the plasmid will die
because they cannot
make adenine. However, under non-selective conditions (the presence of
adenine), cells
which lack the plasmid are able to survive. The YepADE1 plasmid is
unstable because it is
independent of the yeast's chromosomes and is not necessarily evenly
distributed during
mitosis. In a population of dividing cells, some daughter cells may not
receive a copy of the
plasmid. Removing the selective pressure allows survival of those cells
that have lost the
plasmid. Colonies produced from these cells will be red or red sectored.

This exercise demonstrates that
the
maintenance of the white transformant phenotype is dependent on growth
under selective
conditions (MV). One of the white transformant colonies
containing the
YepADE1 plasmid is streaked onto non-selective media, YED. This medium
is
non-selective because it contains adenine.

Day One:

1. Isolate single colonies from one of the
transformant
colonies. Touch
a sterile toothpick to one of the white transformant colonies. Pick a
large white colony
(avoid tiny colonies). Make a streak on a new YED plate. Then use a new sterile toothpick to make another zigzag streak across the
first one on your YED plate.
Continue using fresh sterile toothpicks to make 4 or 5 more zigzag streaks
in this manner. The last streaks should give you some single colonies.
Each colony will grow from a single transformant cell.

2. Follow the same procedure to streak out some of the transformant
cells
on an MV plate. Incubate all plates at 30 C. or room temperature until
single colonies form and their color can be determined.

Day Three or Four: Count the total number of single
colonies on each plate. Count the number of white colonies on each plate.
Use these numbers to compute
the percent of transformants in each plate. Which condition maintained
the highest percentage of transformants? If both YEpADE1 and YCpADE1 were
tested, which was lost at a higher frequency?

Recipes for solutions

Sterile Solutions: prepare working solutions from stock solutions
just prior to use. Use distilled water and autoclave stock solutions and
working solutions.

For additional information

For a very extensive discussion of issues involved in yeast
transformation, see The
Yeast Transformation Homepage maintained by Dr. Dan Gietz of the
University of Manitoba. The protocol described here is based on the
original transformation method of Gietz and Schiestl.